Nitric oxide synthase (NOS) catalyzes the overall conversion of arginine to nitric oxide and citrulline, and proceeds via two partial oxygenation reactions involving the intermediate N-hydroxyarginine (NHA). Ca++/calmodulin and tetrahydrobiopterin (BH4) are required for the overall reaction and for each partial reaction. The enzyme consists of a reductase domain containing NADPH- and flavin-binding sites, and an oxidase domain containing heme and BH4-and arginine-binding sites; the two domains are linked by a calmodulin-binding sequence. The function of BH4 is not clear. We have previously demonstrated with substrate amounts of NOS that the first partial reaction catalyzed by NOS (conversion of arginine to NHA) proceeds in the absence of added electron donor. Thus, the two electrons required for oxygenation of each molecule of arginine to NHA are provided by an enzyme-bound electron donor. Recent studies indicate that NHA formation is approximately stoichiometric with enzyme-bound BH4 oxidation, implicating enzyme-bound BH4 as a source of electrons for NHA synthesis. However, additional studies indicate that another source of electrons is required: 1) NHA synthesis ceases when approximately 70% of the enzyme-bound BH4 remains, 2) NHA synthesis is not proportional to the BH4 content of NOS, 3) NHA synthesis by NOS is stimulated in a dose- dependent manner by the reductase (flavoprotein) domain, but not the oxidase domain. These findings have lead to the novel proposal that enzyme-bound BH4 and reduced flavin (flavin semiquinone) may each be required as a source of electrons for NHA synthesis. Rat brain NOS has been expressed in E.coli, both as the native and histidine-tagged enzyme. Since E.coli is believed to be devoid of BH4, NOS obtained from this source should be free of BH4. Preliminary studies have shown that NOS purified from E.coli surprisingly shows small but significant NOS activity when assayed in the absence of added BH4. We are currently determining whether this small activity represents synthesis of nitric oxide that is independent of BH4 or whether the E.coli enzyme contains bound pterin.